A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease. In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized a knockout alleles of ogt-1 and oga-1 genes. Using a combination of genomic expression arrays and chromatin immunoprecipitation (ChIP) we are looking for genes that respond to nutrient flux differently in the mutants with the hope of identifying pathways of importance. The expression analysis has revealed widespread de-regulation of gene expression in the mutants, identify affected pathways including longevity and aging. We have tested these pathways in the mutants and find alteration in function that are consistent with the gene expression patterns we observe. From the ChIP studies, we have identified a discrete number of genes associated with O-GlcNAcylated proteins. These associations are pronounced at the promoters of the genes and show some overlap with ChIP signals using RNA PolII antibodies. We are currently investigating the function role, if any, of these restricted O-GlcNAc chromatin marks. These marks have the potential to link nutritional flux in the cell directly to gene regulation, offering a novel insight into the role of O-GlcNAc cycling in animal physiology and development. In collaboration with the Hamza Lab (University of Maryland), we have continue to catalog the list of genes regulated by changes in dietary heme concentration using microarray techniques, followed by more in depth studies of individual genes. These results provide insight into heme trafficking pathways that are poorly understood in any biological system. The worm provides a unique means to identify such genes and get information on the biological function of these proteins, some of which are evolutionarily conserved with mammals. In collaboration with the Kostrouch and Kostrouchova Lab (Charles University, Prague) we have continued are long-term studies of the many nuclear hormone receptors (nhrs) in C. elegans. The current year project focused on a set of related nhrs that were found to have interesting responses to nutrient flux. This information adds to our general understanding of nhr function and provides insights into the biological pressures in nematodes that have led to a huge expansion of this class of ligand-regulated transcription factors.